Mitochondria are cytoplasmic organelles that perform many crucial functions in eukaryotic cells, among them generation of most cellular ATP. Mitochondrial dysfunction is implicated in diverse pathologies such as type 2 diabetes, sarcopenia, neurodegeneration, and cancer. Despite their central importance to human health, mechanisms by which mitochondrial functions are regulated remain incompletely understood. The rationale for this application is that improved insights into such mechanisms may permit development of therapeutics to modulate mitochondrial functions as treatments for a wide variety of human diseases. This application focuses on novel roles for sirtuin proteins in regulating key mitochondrial functions. Sirtuins are a family of deacetylases that promote increased longevity in invertebrate models and modulate diverse processes in mammals. The application is based on two novel observations. First, the mitochondrial sirtuin SIRT5 plays a hitherto undescribed role in deacetylating and suppressing activity of Pyruvate Dehydrogenase Complex (PDC), a mitochondrial holoenzyme with a major role in regulating glucose oxidation in mammalian cells. PDC dysfunction is implicated in type 2 diabetes, cancer, and cardiac ischemia. Novel means of stimulating PDC activity - as by SIRT5 inhibition - would be beneficial in these and other clinical settings. Second, the sirtuin SIRT6 has an unexpected role in stimulating mitochondrial respiration. Adipose tissue- specific SIRT6 knockout (S6AKO) mice show marked adiposity, potentially due in part to mitochondrial respiratory defects in brown adipose tissue (BAT). The overall objective of this application is to elucidate novel mechanisms of mitochondrial regulation by sirtuin proteins, thus addressing a key knowledge gap in mitochondrial biology. The hypotheses of this application are two-fold. The first hypothesis is that SIRT5 inhibits glucose oxidation by attenuating PDC activity. The second hypothesis is that SIRT6 promotes mitochondrial respiration to promote cellular and organismal homeostasis. These hypotheses will be tested in two specific aims. First, the roles of SIRT5 in regulating PDC will be elucidated at a mechanistic level through a combination of mass spectrometry, mutagenesis, in vivo flux analysis, and high fat feeding. Second, the role of SIRT6 in promoting mitochondrial respiration will be defined mechanistically. The function of SIRT6 in suppressing adiposity will be elucidated through detailed characterization of S6AKO mice, and through generation of BAT-specific SIRT6 knockouts. This application is innovative, since it focuses on novel functions for sirtuins in regulating mitochondrial energetics. A variety of cutting-edge techniques will be brought to bear to test these hypotheses. The application is significant, since it will establish novel links between sirtuins and mitochondria, potentially laying the groundwork for future sirtuin-directed therapies to modulate glucose oxidation and/or mitochondrial respiration. Hence this work falls within the overall mission of NIGMS.